H3K36me3 Polyclonal Antibody

A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K36me3 (bs-53116R) with peptides containing other H3 and H4 modifications and the unmodified sequence. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure shows a high specificity of the antibody for the modification of interest.

To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3K36me3 (Cat. No. bs-53116R) and the crude serum. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the purified antibody was estimated to be 1:19,300.

ChIP was performed with 2 \u03bcg of the Bioss antibody against H3K36me3 (bs-53116R) on sheared chromatin from 1 million HeLaS3 cells using a ChIP-seq kit. IgG (2 \u03bcg\/IP) was used as a negative IP control. The IP\u2019d DNA was analyzed by qPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the coding region of the active ACTB gene (figure A). The IP\u2019d DNA was subsequently analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure B shows the obtained profiles in genomic regions of chromosome 12 (including the GAPDH positive control), 7 (including the ACTB positive control), 14 and 3, respectively. These results clearly show an enrichment of the H3K36me3 at active genes.

ChIP assays were performed using HeLa cells, the Bioss antibody against H3K36me3 (Cat. No. bs-53116R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1 million cells on the SX-8G IP-Star automated system. A titration consisting of 1, 2, 5 and 10 \u03bcg of antibody per ChIP experiment was analyzed. IgG (2 \u03bcg\/IP) was used as a negative IP control. QPCR was performed with primers for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the Sat2 satellite repeat. The figure shows the recovery expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).